Abstract

Mediators of inflammation, such as PGE2, are known to sensitize the airways to inhaled irritants and circulating autacoids. Evidence from in vivo studies has shown the involvement of vagal pulmonary C-fiber afferents in the PGE2-elicited airway hypersensitivity. However, whether PGE2 acts directly on these sensory nerves is unclear. The present study aimed to investigate whether PGE2 has direct potentiating effects on nodose and jugular pulmonary C neurons cultured from adult Sprague-Dawley rats and, if so, determine whether the EP2 prostanoid receptor is involved. Pulmonary neurons were identified by retrograde labeling with a fluorescent tracer 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate. Using perforated patch-clamp technique, our results showed that1) PGE2 pretreatment (1 μM) increased the whole cell current density elicited by capsaicin and phenylbiguanide, chemical agents known to stimulate pulmonary C fibers; 2) selective activation of the EP2 prostanoid receptor by butaprost (3–10 μM) increased the whole cell current density elicited by capsaicin; and 3) PGE2, as well as butaprost, increased the number of action potentials evoked by current injection. Therefore, we conclude that PGE2 directly sensitizes vagal pulmonary C neurons to chemical and electrical stimulation. Furthermore, butaprost modulates the neurons in a manner similar to that of PGE2, suggesting that the effects of PGE2 are mediated, at least in part, through the EP2 prostanoid receptor.

EP2 prostanoid receptor

butaprost

1,1′-dioctadecyl-3,3, 3′,3′-tetramethylindocarbocyanine perchlorate

nodose ganglion

jugular ganglion

during inflammatory reaction of the airways, various autacoids (e.g., histamine, thromboxanes, and prostaglandins) are locally released by a variety of cells, including airway epithelia, leukocytes, and mast cells. PGE2 is a particularly abundant prostanoid found in the lungs and airways during inflammation. For example, PGE2levels in the bronchoalveolar lavage of human patients can increase 2- to 10-fold during an asthmatic episode (18, 22, 28). Furthermore, in guinea pig nodose ganglia, ovalbumin sensitization can elicit a fivefold increase in the release of PGE2 from mast cells within the ganglia (37).

Once released, PGE2 binds to specific prostanoid receptors on target cells and is then rapidly removed by prostaglandin transporters on a variety of cells (33). To date, four subtypes of prostanoid receptors that exhibit the highest affinity to PGE2 are known: EP1, EP2, EP3, and EP4 (10, 31). All are members of the superfamily of G protein-coupled receptors that possess a seven-transmembrane domain topology and are linked to heterotrimeric G protein molecules. The subtypes differ in their distribution in various tissues and signal transduction mechanism (31). The prostaglandin receptors EP2, EP3C, and EP4 are coupled to Gs protein and provoke increases in cAMP (31). Studies in dorsal root ganglion nociceptive neurons have shown that the sensitizing effects of PGE2 are mediated through a cAMP-protein kinase A signal transduction mechanism, suggesting that activation of the EP2, EP3C, or EP4 receptor may be involved (12, 24, 29, 34).

The lungs and airways are extensively innervated by vagal sensory fibers that are sensitive to circulating autacoids and inhaled irritants. The foremost of these chemosensitive neurons innervating the respiratory tract is the C-fiber afferent (2). The somata of these neurons reside in the nodose and jugular ganglia and send projections to the nucleus tractus solitarius, thus relaying information from the lungs and airways to medullary respiratory centers. The primary function of these neurons under normal physiological conditions is protective: when the sensory terminals are activated by irritants, cholinergic and tachykininergic reflexes are initiated, resulting in bronchoconstriction, mucous secretion, and cough, which limit further infiltration of the irritants deeper into the airways. Under certain pathological states, however, these afferent nerves develop hypersensitivity, and the exaggerated reflex responses become deleterious to normal airway function. For example, during airway inflammatory reaction (e.g., in asthma), the airways become exquisitely sensitive, and irritants can elicit a far more severe bronchoconstriction and more copious mucous secretion.

A previous study in our laboratory showed that PGE2infusion markedly enhances the apneic response elicited by chemical stimuli in intact rats (26). A subsequent study demonstrated that PGE2 increased the single-unit vagal C-fiber activity elicited by capsaicin and other chemical stimulants (16). These two studies clearly showed that PGE2 enhances the sensitivity of vagal C fibers. However, the effects of PGE2 on other types of cells (e.g., smooth muscles) in the lung are well documented, and whether PGE2acts directly on the afferent fiber is unclear. In addition, the subtype of prostanoid receptor mediating these potentiating effects is unknown. Therefore, the purpose of this study was to determine, in acutely isolated cultured neurons, whether PGE2 could directly modulate the function of vagal bronchopulmonary C neurons. Furthermore, we hypothesized that selective activation of the EP2 prostanoid receptor could sensitize the vagal pulmonary neurons; the availability of a selective agonist of the EP2receptor, butaprost, made it feasible to test this hypothesis (23).

METHODS

Labeling respiratory vagal neurons with DiI.

Sensory neurons innervating the lungs and airways were identified by retrograde labeling from the lungs by using the fluorescent tracer, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI). Young adult (150–200 g) male Sprague-Dawley rats were anesthetized with an intraperitoneal injection of pentobarbital (50–60 mg/kg) and intubated with a polyethylene tube such that the tip rested in the trachea above the thoracic inlet. With the rat tilted head up at ∼30°, DiI (0.25 mg/ml; 1% ethanol concentration) was initially dissolved and sonicated in ethanol, diluted in saline, and then instilled into the lungs (2 × 0.25 ml). After 7–11 days, an interval previously determined to be sufficient for the dye to diffuse to the cell body, the nodose and jugular ganglia were extracted.

Cell culture.

The animals were anesthetized with halothane (100%) and immediately decapitated. The head was immersed in ice-cold Hanks' balanced salt solution. Within 10 min, nodose and jugular ganglia with attached nerve trunks were extracted under a dissecting microscope and placed in ice-cold DMEM-F12 solution.

Nodose ganglia were separated from jugular ganglia and cultured separately. Each ganglion was desheathed, cut into ∼10 pieces, placed in 0.125% type IV collagenase, and incubated in a humidified chamber for 1 h in 5% CO2 in air at 37°C. The ganglion suspension was centrifuged (150 g, 5 min), and the supernatant was aspirated. The ganglion pellet was briefly (<1 min) resuspended in 0.05% trypsin and 0.53 mM EDTA in Hanks' balanced salt solution and centrifuged (150 g, 5 min). The ganglion pellet was then resuspended in a modified DMEM-F12 solution [DMEM-F12 supplemented with 10% (vol/vol) heat-inactivated fetal bovine serum, 100 U/ml penicillin, 100 μg/ml streptomycin, and 100 μM MEM nonessential amino acids] and gently titrated with a small-bore fire-polished Pasteur pipette. The dispersed cell suspension was centrifuged (500 g, 8 min) through a layer of 15% bovine serum albumin to separate the cells from the myelin debris. The pellets of nodose and jugular ganglion cells were resuspended in the modified DMEM-F12 solution supplemented with 50 ng/ml 2.5S nerve growth factor (NGF) and plated onto dry poly-l-lysine-coated glass coverslips. Initially, small droplets of resuspended cells were incubated to promote cell adhesion at a high density. After 3 h, the coverslips were immersed in the modified DMEM-F12-NGF media and incubated overnight (5% CO2 in air at 37°C).

Electrophysiological recording.

Borosilicate glass was pulled by using a micropipette puller and fire polished to a tip resistance of 2–4 MΩ. The micropipettes were coated with Sylgard (Dow Corning) to minimize pipette capacitance. The micropipette electrode was a silver-silver chloride electrode. The silver-silver chloride pellet reference electrode was placed in a dish of pipette solution, and a 1% agar salt bridge closed the circuit to the bath solution. The liquid junction potentials and agar bridge and bath potentials were symmetrical and produced opposing junction effects. Calculations for liquid junction potential were performed by using the junction potential module within the data-acquisition software, based on JPCalc software (5).

Electrophysiological recordings were made in a small-volume (0.15 ml) perfusion chamber at room temperature. The coverslip containing the attached cells was centered in the perfusion chamber, which was perfused by gravity feed with PGE2, butaprost, or vehicle (extracellular solution) at ∼1 ml/min, while various chemical stimulants were delivered by a three-channel fast-stepping perfusion system, with its tip positioned to ensure that the cell was fully within the stream of the perfusate. Two chemical stimulants were chosen for this study: capsaicin and phenylbiguanide. The former is a potent stimulant of C-fiber afferents and a selective agonist of the type 1 vanilloid receptor (6), and the latter is a selective agonist of the type 3 5-hydroxytryptamine (5-HT3) ionotropic receptor, a ligand-gated cation channel (30). 5-HT (5-hydroxytryptamine or serotonin) is a potent inflammatory mediator in the airways. Drugs were dissolved in extracellular solution and adjusted to an osmolarity of ∼310 mosM and pH of 7.4. Data were collected from only one cell per dish to avoid possible contamination of the cells by the chemical agents. Cells harvested from a single animal were divided among different experimental protocols.

For all electrophysiological experiments, we selected single cells for analysis that met the following criteria: 1) spherical shape with no neurite outgrowths, 2) small size (diameter <35 μm), 3) activated by either capsaicin or phenylbiguanide,4) labeled with DiI fluorescence, and 5) quiescent with a stable resting membrane potential more negative than −40 mV.

Patch-clamp recordings were made in the whole cell perforated patch configuration (gramicidin 50 μg/ml) by using Axopatch 200B/pCLAMP8 (Axon Instruments, Foster City, CA). Responses of these neurons to capsaicin and phenylbiguanide are relatively slow; therefore, the signals were filtered at 2 or 5 kHz and digitized at 5 or 10 kHz. Series resistance (4–10 MΩ) was compensated at ∼80%. Input resistance was determined by applying a series of small hyperpolarizing current pulses in 10-pA increments of 280-ms duration from 10 to 100 pA. The input resistance was taken as the slope of the linear portion of the plot of the mean steady-state voltage vs. the command current pulses.

Membrane depolarization and action potential generation by chemical and electrical stimulation were recorded in current-clamp mode. For electrical stimulation, current injections were applied in 10-pA increments of 280-ms duration from 0 to 100 pA. Whole cell currents evoked by ligands and voltage steps were obtained in voltage-clamp mode from a holding potential of −60 mV. Voltage steps were applied in 5-mV increments of 50-ms duration to test potentials between −80 and +75 mV. The signals were filtered at 10 kHz and digitized at 100 kHz.

Voltage-gated currents were analyzed quantitatively by fitting activation curves to a modified Boltzmann equation:I(V) =Gmax × (V −Erev) × {1 + exp[(V½ −V)/k]}−1, whereI(V) is the current generated at the test potential, Gmax is the maximum macroscopic conductance, V is the test potential,Erev is the reversal potential (the zero-current potential), V½ is the half-activation voltage (voltage at which one-half of the maximal conductance is achieved), andk is the slope factor (the slope of the linear portion of the conductance-voltage relationship). The Boltzmann equation describes the probability of channels being open or closed as a function of voltage (15).

Experimental design.

The experiments were carried out in two study series. The first series aimed to test whether PGE2 pretreatment modulates the sensitivity of cultured pulmonary C neurons to various chemical and electrical stimuli. The second series aimed to determine the effect of selective activation of the EP2 prostanoid receptor on the sensitivity of cultured C neurons. The stimulus was applied before and then after pretreatment of the cell with either PGE2, butaprost, or vehicle for 5 min and after a 1-min washout. In some cells, the recovery responses were studied after washout of PGE2 or butaprost for >20 min. Although each neuron was identified as originating from either the nodose or jugular ganglion, there was no detectable difference in the responses between the two populations; their data were pooled for statistical analysis.

Data are reported as means ± SE. Statistical comparisons between control and treatment conditions were evaluated by using a single-factor ANOVA with repeated measures. A P value < 0.05 was considered significant.

RESULTS

DiI-labeled vagal bronchopulmonary neurons.

The composite photomicrograph in Fig. 1represents the distribution of DiI fluorescence in the respiratory tract and the selective labeling of pulmonary neurons cultured from the vagal ganglia. The airway epithelium of the trachea showed prominent DiI fluorescence (Fig. 1A), which was evenly distributed throughout the lung, including bronchioles and alveoli (Fig.1B). Because the jugular and nodose ganglia innervate multiple visceral organs, we examined a variety of tissues in the nervous and other organ systems, besides the lungs and trachea, after they were fixated with 4% paraformaldehyde and sliced in ∼50-μm sections, for possible contamination by the DiI labeling: spinal cord and dorsal root ganglia (thoracic and lumbar), heart, blood, kidney, liver, esophagus, stomach, and intestine. DiI fluorescence was found only in nodose (e.g., Fig. 1C) and jugular ganglia, lungs, and airways. Because DiI labeling was sequestered in the lungs and airways, the labeled nodose and jugular ganglion cells represented neurons selectively innervating the respiratory tract. We found that 10.6% of the cultured nodose ganglion cells and 2.9% of the jugular ganglion cells were labeled.

In two separate groups of rats (n = 7 each) prepared in a manner identical to that described in this study, we found that there is no difference between control and DiI-labeled rats in the sensitivity of the pulmonary chemoreflex responses (apnea, bradycardia, and hypotension) elicited by pulmonary C-fiber stimulation with either capsaicin or phenylbiguanide, indicating that DiI-labeling did not alter the normal functions of these neurons (K. Kwong, J. Parsons, and L.-Y. Lee, unpublished observation).

In voltage-clamp mode, in pulmonary nodose and jugular ganglion neurons, capsaicin in the range of 0.01–0.3 μM elicited incremental whole cell currents in a dose-dependent manner (e.g., Fig.2A). PGE2pretreatment increased both the peak and the duration of the inward current response elicited by capsaicin; an example is shown in Fig.2B. In this neuron, the potentiating effects of PGE2 recovered within 15 min, and both the peak and duration of the response returned to control levels. Whereas the vehicle for PGE2 had no effect on the cellular response to capsaicin (Fig. 2D), PGE2 pretreatment increased the whole cell current density elicited by capsaicin over that of control in a separate group of neurons (Fig. 2D; control: −23.1 ± 7.5 pA/pF; PGE2: −42.4 ± 14.8 pA/pF;P < 0.05; n = 7). In four of these neurons, we were able to maintain the gigaohm seal long enough to record a recovery from the PGE2 pretreatment (control: −33.8 ± 9.4 pA/pF; recovery: −31.6 ± 8.2 pA/pF;P > 0.05). A representative sample of pulmonary vagal neurons displayed an average resting membrane potential of −60.8 ± 1.3 mV and a whole cell capacitance of 17.9 ± 1.0 pF (n = 30) under normal ionic conditions; the latter was directly proportional to the surface area of the cell membrane (15) and was measured electronically with the Axon 200B amplifier.

PGE2 pretreatment potentiates the whole cell inward current elicited by chemical stimuli. A: in voltage-clamp mode (Vhold = −60 mV), capsaicin (Cap) delivered at varying doses elicited whole cell inward currents in a dose-dependent manner in a small pulmonary jugular ganglion neuron (17.4 pF). Bar, duration of Cap challenge, 4 s; 10-min interval between these and all subsequent challenges.B: experimental record in a small pulmonary nodose ganglion neuron (14.4 pF) showing the response to Cap (0.1 μM, 4 s) was potentiated after PGE2 pretreatment (1 μM, 5 min;middle trace). In this neuron, the gigaohm seal was maintained long enough to record a recovery response (righttrace; ∼15 min after termination of PGE2 perfusion) that was similar to that of control (Con) levels (left trace).C: in a different pulmonary nodose neuron (14.6 pF), the same dose of PGE2 potentiated the inward current elicited by phenylbiguanide (PBG; 1 μM, 4 s; right trace). Recovery was not tested in this neuron. D: left: responses of individual neurons (lines) and group means (circles) of the whole cell current densities elicited by Cap (0.1 μM, 4 s) before (Con) and after PGE2 pretreatment (1 μM, 5 min). * Significantly different from Con (P < 0.05;n = 7). Right: pretreatment with the vehicle (Veh) for PGE2 did not alter the response to the same dose of Cap in a separate group of neurons (P > 0.05;n = 7).

To determine whether PGE2 pretreatment could also potentiate the responses of other ligand-gated channels, we tested the whole cell current response to phenylbiguanide; the response was also potentiated by the same dose of PGE2 (e.g., Fig.2C). This finding is consistent with the results reported in our recent study (13), wherein PGE2 induced a similar and reversible potentiating effect on the response to phenylbiguanide in cultured rat nodose and jugular C neurons.

In current-clamp mode, PGE2 pretreatment (1 μM, 5 min) increased the number of action potentials elicited by capsaicin (e.g., Fig. 3A; 0.1 μM, 4 s). Furthermore, PGE2 decreased the onset latency of the action potentials; the subthreshold depolarization was greater in magnitude and duration. At higher depolarized membrane potentials, the magnitude of the action potentials progressively diminished and eventually ceased, presumably because of a progressive inactivation of voltage-gated sodium channels. The potentiated responses induced by PGE2 may, therefore, be underestimated by measurement of the number of action potentials alone. Moreover, the magnitude of the capsaicin-evoked depolarization varied substantially among different neurons (e.g., Fig. 3, C and D). Nonetheless, PGE2 pretreatment tended to increase the number of action potentials (increase = 84.4 ± 36.7%; P > 0.05) in response to the same capsaicin challenge; the potentiated response was observed in four out of five neurons tested (Fig.3C). After 30–120 min, the potentiated responses were almost fully recovered in the three cells that were tested (Fig. 3, A and C).

PGE2 and butaprost (Buta) enhance the no. of Cap-evoked action potentials. A: experimental record in current-clamp mode shows that pretreatment with PGE2 (1 μM, 5 min) increased the no. of action potentials elicited by Cap (0.1 μM, 4 s; middle trace) in an isolated cultured pulmonary nodose ganglion neuron (18.0 pF). The response during recovery (right trace) returned toward that of Con (left trace) after ∼2 h. B: pretreatment with Buta (3 μM, 5 min) also potentiated the response evoked by Cap (0.1 μM, 4 s; middle trace) over that of Con (left trace) in a pulmonary nodose neuron (19.4 pF). In this cell, the recovery response (right trace) returned toward Con levels after ∼45 min. C: composite graph showing that the response to Cap (0.1 μM, 4 s) increased in 4 out of 5 pulmonary vagal ganglion neurons (lines) after PGE2pretreatment (1 μM, 5 min) compared with that of Con. The responses in all 3 neurons tested for recovery (Rec) returned toward Con levels 30–120 min later. D: in 5 other pulmonary vagal ganglion neurons (lines), the response to the same dose of Cap increased after Buta pretreatment (3–10 μM, 5 min) in 4 of them; all 3 of the neurons tested for a Rec response returned completely to that of Con levels.

In addition to ligand-gated currents, PGE2 could also modulate voltage-gated channels. We applied a current injection protocol (0–100 pA, inward; 10-pA increase; 280 ms) to test this possibility. Figure 4A is a representative experiment record from a small-diameter DiI-labeled nodose neuron, depicting how PGE2 increased the number of action potentials elicited by three different magnitudes of current injection. During recovery (∼1 h), the number of action potentials elicited by all three current injection levels returned toward control levels. On average, PGE2significantly increased the number of action potentials elicited at these three current injection levels over that of control (Fig.4B).

Because an increase in Na+ (or Ca2+) currents or a decrease in outward K+ currents may contribute to an increase in action potentials, we tested whether PGE2changes the active membrane properties of these neurons. In reduced-Na+ extracellular solution, the step potentials of the voltage-clamp protocol evoked a transient inward current and a sustained outward current (Fig.5A). We analyzed the transient inward currents (Fig. 5B) by fitting the activation curves to a modified Boltzmann equation; an example is shown in Fig.5C. After PGE2 pretreatment, there was a hyperpolarizing shift in the half-activation potential and a steeper magnitude of the k slope factor (Fig. 5D); the former indicates that channels are opened at a smaller membrane depolarization, and the latter suggests that the channels are more sensitive to voltage changes. We failed to detect a change inErev and Gmax, indicating that neither ion selectivity nor the maximum number of participating ion channels has changed. We then examined whether outward currents were modulated by the PGE2. In the same four cells, only one neuron showed a decrease in the steady-state outward conductance after PGE2 pretreatment. The input resistance did not change after PGE2 pretreatment (control: 387.9 ± 59.0 MΩ; PGE2: 478.4 ± 86.3 MΩ;P > 0.05; n = 9).

Buta enhances the whole cell inward current elicited by Cap. A: selective activation of the EP2prostanoid receptor by Buta (3 μM; 5 min) caused an increase in the whole cell current response to Cap (0.1 μM, 4 s;Vhold = −60 mV; middle trace) in a pulmonary nodose ganglion neuron (26.6 pF). The response during recovery (right trace) was similar to that of Con (left trace). B: group data from both nodose and jugular ganglion neurons showing the significant increase in the Cap-evoked whole cell current density after Buta pretreatment (3–10 μM, 5 min) compared with that of Con. Lines, responses from individual neurons; ●, group means. * Significantly different from Con (P < 0.05; n = 5).

To determine whether activation of the EP2 receptor could also modulate the voltage-gated channels in pulmonary C neurons, we tested the response to electrical stimulation. Butaprost pretreatment significantly increased the number of action potentials evoked by the medium current injection level (P < 0.05;n = 10). Butaprost pretreatment also showed a trend of increasing the number of action potentials evoked by the low- and high-current injections (Fig. 7), but the increases were not statistically significant.

Buta sensitizes pulmonary vagal ganglion neurons to electrical stimulation. A: with the use of the identical current injection protocol as in Fig. 4, Buta pretreatment (3 μM, 5 min) increased the no. of action potentials evoked at the 2 higher levels of current injection (bar, 280 ms; bottom) compared with that of Con (top) in a pulmonary jugular neuron (12.1 pF). Recovery was not tested in this neuron. B: group data reveal that, after Buta pretreatment (3–10 μM, 5 min), the no. of action potentials elicited by the medium current injection (70 pA) increased compared with that of Con in vagal pulmonary neurons. Dotted lines, responses of individual neurons; solid circles, group means. * Significantly different from Con (P < 0.05;n = 10). See Fig. 4 legend for further explanation.

DISCUSSION

Our study showed that, in cultured adult rat pulmonary vagal C neurons, PGE2 potentiates the response to chemical and electrical stimuli. This potentiation is probably mediated, at least in part, through the EP2 prostanoid receptor. Our results clearly demonstrate that PGE2 and butaprost exert direct sensitizing effects on cultured vagal bronchopulmonary C neurons. Specifically, in voltage-clamp mode, PGE2 increased the whole cell current density evoked by capsaicin. In current-clamp mode, PGE2 increased the number of action potentials elicited by capsaicin and current injection. Very similar responses were observed when the EP2 prostanoid receptor was selectively activated by butaprost. Together, these data suggest that activation of the EP2 receptor, either by PGE2 or butaprost, has the ability to modulate the function of both ligand-gated and voltage-gated ion channels in isolated pulmonary chemosensitive neurons.

To our knowledge, this is the first report of the direct sensitizing effects of PGE2 and butaprost in isolated vagal neurons innervating the lungs and airways. Our results are consistent with and build on previous experiments that showed PGE2-induced potentiation of the pulmonary chemoreflex response and the single-unit C-fiber sensitivity (16, 26). In those studies in intact animals, we were unable to determine whether PGE2 was acting on C fibers directly or through an intermediary effect on other cells (e.g., degranulation of mast cells). We should point out that these direct and indirect actions of PGE2 are not mutually exclusive. In addition, we are making electrophysiological recordings from the perikaryon with the assumption that the channels, receptors, and intracellular signaling mechanisms present on the nerve terminals are also present on the cell body. However, it is possible that functional expression of these proteins is not present or is present at a significantly lower density in the soma. Therefore, the current densities elicited by capsaicin recorded at the cell bodies may not exactly match those elicited at the terminals. Furthermore, the magnitude of elicited currents that would have evoked action potentials at the sensory terminal may not evoke them at the cell soma. In addition, because we were recording the responses of these neurons at the temperature and partial pressures of O2 and CO2 of room air, the magnitude of the responses may not be the same as that of in vivo neurons. Despite these potential limitations, our data demonstrate expression of functional capsaicin, 5-HT3, and EP2 receptors on the cell body and a distinct sensitizing effect of PGE2 on the isolated neurons.

The fact that butaprost also enhances the excitability of pulmonary C neurons (Figs. 3, 6, and 7), in a manner similar to that observed with PGE2, suggests that PGE2 may act through the EP2 receptor in these cultured neurons. Butaprost is highly selective for the EP2 prostanoid receptor subtype and does not bind to EP1, EP3, or EP4prostanoid receptors at the concentrations used in this study (23). However, definitive tests ultimately confirming the involvement of the EP2 receptor in pulmonary C neurons require the use of a selective EP2-receptor antagonist, which is, however, presently not yet available. Whether other prostanoid-receptor subtypes (either singly or in combination) play a role in modulating pulmonary C neurons is not known. It is possible that other members of the EPx family could also modulate the sensitivity of these neurons, such as that suggested in dorsal root ganglion neurons with the EP3C and EP4receptors (35). Additionally, perhaps other classes of prostanoid receptors coexpress and interact. For example, the prostacyclin receptor (IP) mRNA is coexpressed with EP1, EP3, or EP4 mRNA in some dorsal root ganglion neurons (32). The potential contribution of other EPx receptors in modulation of pulmonary C neurons should be further explored when selective pharmacological agents become available.

This study revealed that activation of the EP2 receptor can modulate the function of a capsaicin receptor, the 5-HT3receptor, and one or more voltage-gated ion channels. The effects of EP2-receptor activation are thought to be mediated through activation of protein kinase A. In a parallel study measuring calcium transients with fura 2 in cultured vagal chemosensitive neurons, Gu et al. (13) found that forskolin mimicked the potentiating effects of PGE2, whereas H89, a membrane-permeant inhibitor of protein kinase A, inhibited them. Possible end-targets of EP2 activation include type 1 vanilloid receptor, which has been identified on nodose neurons and has several predicted protein kinase A phosphorylation sites; TTX-resistant and TTX-sensitive sodium channels (e.g., SNS/PN3); calcium-dependent potassium currents (e.g., SK); and hyperpolarization-activated cation current (3, 6, 14,20, 21, 38). It is likely that activation of the EP2receptor by PGE2 results in the protein kinase A-mediated phosphorylation of both ligand-gated and voltage-sensitive channels, thus increasing their probability of opening (15).

Our results clearly demonstrate the labeling of neurons innervating the bronchopulmonary region by DiI; we did not detect the fluorescent dye in any of the tissues outside of the pulmonary regions. In contrast, the dye coverage throughout the respiratory tree was extensive: we found labeling from trachea to alveoli and in all lobes of the lung. When pooled, both vagal ganglia showed 7.3% of the cells labeled. This figure is slightly lower than that reported in the cat, which revealed that ∼15% of vagal afferent fibers innervate the respiratory tree (2), and higher than the ∼4% of nodose ganglion neurons that innervate the guinea pig trachea (8). The discrepancies could be due to species difference, or, alternatively, it is possible that part of the lungs and nerve endings may not have been exposed to a sufficient amount of DiI, and, therefore, the number of pulmonary neurons is underestimated. When we analyzed the nodose and jugular ganglion separately, we were surprised to find a fivefold increase in the percentage of labeled nodose neurons vs. jugular neurons. In contrast, Hunter and Undem (19) found that the guinea pig tracheal epithelium, when labeled with rhodamine microspheres, receives sensory innervation almost exclusively from the jugular ganglia. That study found that, when fast blue dye was used, which is able to penetrate and label nerves innervating deeper structures (e.g., submucosa), the dye was more evenly distributed between the two ganglia. It is also possible that, in the rat, the more distal regions of the respiratory tree receive innervation from a larger percentage of nodose ganglion neurons relative to that of the jugular ganglion neurons. Therefore, the differences between our two studies could be due to a combination of species difference and a difference in the distribution of sensory innervation between trachea and lung periphery. It must be noted that, despite these quantitative differences in labeling percentage, the precision with which respiratory structures are identified remains exceedingly high in this study.

In these experiments, we selected small-diameter neurons for study. It has been shown that the smaller diameter cultured isolated neurons correspond to the substance P-containing, neurofilament-negative, chemosensitive C fibers (9, 39). However, we cannot rule out the possibility that a fraction of the chemosensitive cells that we studied is in fact Aδ-neurons. Aδ-afferents are a heterogeneous population of thin myelinated fibers, a subset of which is characterized by their polymodal sensitivities and chemosensitive properties (17, 25). Although these Aδ-neurons share some similarities in their physiological properties with C neurons, the proportion of capsaicin-sensitive pulmonary Aδ-fibers to C fibers is miniscule in the rat, according to the study by Ho and coworkers (17). Therefore, although Aδ-neurons may be present in the population of small chemosensitive neurons that we studied, the vast majority of them are C neurons.

The pulmonary C-fiber afferents exhibit polymodal sensitivities: known stimulators include capsaicin, bradykinin, histamine, hypertonic saline, adenosine, adenosine triphosphate, acid, heat, wood smoke, and mechanical stimuli (11, 27). Indeed, receptors for these compounds have been identified on various C-fiber neurons across species, including ferret, rabbit, guinea pig, rat, and humans (11, 27). These C-fiber terminals have extensive innervation throughout the respiratory tree. A dense plexus of nerve terminals exists below and between the lung and airway epithelial cells, in close proximity to the airway lumen (1, 4). From such a vantage point, these nerve terminals are able to monitor the airways for inhaled irritants as well as local and circulating autacoids. During inflammation, many circulating and locally released autacoids, particularly PGE2, sensitize these C fibers, which lead to an exaggerated bronchopulmonary reflex response. In healthy human subjects, inhalation of aerosolized PGE2elicits the sensation of dyspnea during exercise and enhances the cough reflex elicited by capsaicin and phenylbiguanide (7,36). Results of this study have established direct evidence of the PGE2-induced hypersensitivity of bronchopulmonary C neurons, lending further support to these previous studies.

In summary, data from this study clearly show that PGE2enhances the excitability of cultured pulmonary vagal chemosensitive neurons to both chemical and electrical stimuli. This study has, therefore, provided direct evidence in support of the previous finding of a sensitizing effect of PGE2 on vagal pulmonary C-fiber afferents in vivo. These results further reveal that the sensitizing effect of PGE2 is probably mediated, at least in part, through the activation of the EP2 prostanoid receptor, which, in turn, modulates the functions of a variety of ligand-gated and voltage-sensitive ion channels on the neuronal somata.

Acknowledgments

The authors are indebted to Lifang Zhang, Robert F. Morton, Jeremy Parsons, Cynthia Long, Dr. Qihai Gu, and Dr. You Shuei Lin for technical assistance in this study. The authors also thank Dr. Jonathan Satin for helpful suggestions and comments on the manuscript.

Footnotes

This study was supported by National Heart, Lung, and Blood Institute Grants HL-58686 and HL-67379.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.